This project studies cellular and physiological aspects of neuronal calcium (Ca2+) signaling, with long-range emphasis on postsynaptic responses in central nervous system neurons. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca2+ concentration that is strongly modulated by the transport activity of intracellular calcium storage organelles. This transport activity plays an important role in spatio-temporally shaping the signals that regulate downstream processes like gene expression and synaptic plasticity. Several labs, including ours, had earlier shown in a variety of neurons that stimulus-evoked cytosolic free Ca2+ elevations induce large, reversible increases in the concentration of calcium within mitochondria, which in turn has important effects on physiological and pathophysiological processes. This year we continued to explore the consequences of evoked mitochondrial Ca2+ uptake, with emphasis on understanding the mechanisms of ischemic preconditioning (PC), an important but poorly understood phenomenon whereby neurons become tolerant to a normally lethal insult after pretreatment with a similar but milder, non-lethal challenge.[unreadable] It was previously shown that NMDA overstimulation that leads to excitotoxic delayed cell death (DCD) is associated with strong mitochondrial Ca2+ uptake. This mitochondrial activity is spatially heterogeneous, which has proven to be important because the number and location of damaged mitochondria determine a cell?s vulnerability to excessive NMDA. This year we have explored the hypothesis that the reduced mitochondrial dysfunction accounts for the neuroprotective effects of ischemic preconditioning. The results from several standard preconditioning protocols showed that at the cellular level these treatments qualitatively recapitulate the effects of NMDA by inducing the redistribution of intracellular Ca2+ and the transient Ca2+ loading and depolarization of mitochondria. However, these effects were weaker, fully reversible and did not lead to DCD. Certain protocols induced stronger neuroprotection than others but, regardless of efficacy, PC neuroprotection was paralleled by reduced mitochondrial injury after excitotoxic NMDA exposure. In addition, stronger PC also substantially reduced Ca2+ entry. These results indicate that PC exerts a protective effect by increasing mitochondrial tolerance for large Ca loads, thereby attenuating mitochondrial dysfunction. The most effective PC protocols appear to recruit additional, additive mechanisms. One such mechanism uncovered this year involves a significant down-regulation of surface-expressed, and therefore active, NMDA receptors.[unreadable] Slice cultures of hippocampus represent an alternative model for studying neuronal tolerance, since pyramidal neurons of the CA1 region are quite sensitive to excitotoxic stimuli, while neurons in the CA3 region show a high level of endogenous neuroprotection. Consistent with previous evidence that mitochondrial damage is a key event in determining excitotoxic vulnerability, the fraction of swollen, damaged mitochondria in CA1 dendrites following application of excitotoxic NMDA was essentially 100%, while in CA3 only a subset of mitochondria was swollen, the rest retaining their normal, rod-like shape. In cell bodies of both regions, many mitochondria were damaged, but again these were more numerous in CA1 cells. Results suggest that mitochondrial damage is a major factor in the selective vulnerability of CA1 neurons to excitotoxic insult, which in turn underlies their susceptibility to ischemic injury, and justify further investigation into how the mitochondria of CA3 differ from those of CA1.